Yeast cells undergo periodic fluctuations in density during the cell division cycle such that a minimum in density occurs at the time of cell separation whereas a maximum occurs between the time of deoxyribonucleic acid replication and nuclear division. Synchronous cultures can be selected from asynchronously growing cell cultures by withdrawing the cells of least or greatest density after banding in Renografin-sucrose density gradients. This technique is rapid, reproducible, and almost

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... d in capacity. Two methods have been utilized to obtain synchronous cultures of yeast cells. The first discovered by Williamson and Scopes (8) takes advantage of the fact that certain strains of yeast collect in the stationary phase as unbudded cells which, upon reinoculation into fresh growth medium, initiate budding synchronously. The second, developed by Mitchison and Vincent (3), involves the selection of unbudded cells from a growing population by differential sucrose density gradient sedimentation. Our analysis of temperature-sensitive yeast mutants defective in specific steps of the cell division cycle (1) requires the use of synchronous cultures, but both of the previously described methods have certain disadvantages. First, both methods are strain dependent. In addition, the method of Williamson and Scopes utilizes cells that are adapting from the stationary to the growth phase, and results obtained with such cells cannot unequivocally be assumed to be valid for cells in the exponential growth phase. The method of Mitchison and Vincent overcomes this objection since synchronous cells are selected from an exponentially growing asynchronous population; however, the necessity of layering a cell suspension on a sucrose gradient limits one to the use of relatively small numbers of cells to avoid "streaming" in the gradient. I attempted, therefore, to find a technique which would overcome both objections by allowing the selection of cells from an exponentially growing asynchronous population in relatively large numbers. The technique to be described herein utilizes the finding that yeast cells undergo periodic fluctuations in density during the cell division cycle by selecting synchronous cells after isopycnic equilibrium banding in Renografin-sucrose density gradients. Equilibrium density centrifugation has recently been reported to be effective in selecting synchronous cell populations of Chlorella (6). MATERIALS AND METHODS Media and strains. Cells were grown in synthetic medium [6.7 g of yeast nitrogen base (Difco), 10 g of succinic acid, 6 g of NaOH, 2 g of glucose, 0.04 g of tyrosine, 0.04 g of lysine, 0.04 g of histidine, 0.01 g of adenine, and 0.01 g of uracil, in 1 liter of water at a final pH level of 5.8] with the addition of 0.1% yeast extract. The strain of Saccharomyces cerevisiae used in these experiments, designated A364A D-5 (a/a adel/adel ural/ural tyrl/tyri his7/his7 lys2/ + leu2/ + gall/gall), is a diploid resulting from the mating of haploid segregants from the cross, A364A (a adel ade2 ural his7 lys2 gall) X 79.20.3 (a adel (2) ural ku2 gall). Isopycnic banding. In the experiments reported here, an exponentially growing, asynchronous population of cells was collected by centrifugation, chilled, and suspended in 7 ml of a solution containing 20% Renografin (E. R. Squibb & Sons, Inc., New York) by weight. A continuous density gradient was then formed by using this suspension as the light component and a 7-ml solution containing 20% Renografin plus 20% sucrose by weight as the dense component in a 15-ml glass centrifuge tube. The gradient was then centrifuged at 12,000 rev/min for 1 to 2 min in a Sorvall HB4 rotor at 5 C. Longer periods of centrifugation are not advisable since the cells slowly take up sucrose and migrate to the bottom of the tube. Three to five per cent of the top-most or bottom-most cells were then removed from the gradient and inoculated directly into growth media. More recently, we have been using a gradient of 20 to 27% Renografin containing 1% polyvinyl pyrollidone (PVP 360). Under the latter conditions true equilibrium is at-1280 on May 9, 2020 by guest